Leptospira interrogans Outer Membrane Protein-Based Nanohybrid Sensor for the Diagnosis of Leptospirosis
<p>Characterization of the DNA sensor fabrication steps using a Fourier-transform infrared spectroscopy (FTIR) analysis of (<b>A</b>) the AuN/CNF bare electrode and (<b>B</b>) after modifications of the AuN/CNF electrode surface with a single stranded DNA (ssDNA) probe (AuN/CNF/ssDNA<sub>probe</sub>) at a frequency scan of 500–3500 cm<sup>−1</sup>.</p> "> Figure 2
<p>Raman spectrum of (<b>A</b>) the bare AuN/CNF electrode and (<b>B</b>) 5′amino-linked ssDNA probe-modified AuN/CNF electrode (AuN/CNF/ssDNA<sub>(probe)</sub>).</p> "> Figure 3
<p>Voltammetric analysis of the developed DNA sensor in different phases of the fabrication, including (A) the AuN/CNF electrode (bare), (B) AuN/CNF/ssDNA (probe), and (C–O) hybridization with single-stranded GDNA of <span class="html-italic">Leptospira interrogans</span>. The insert (<b>I</b>) shows a linear curve for the calculation of the limit of detection (LOD) and (<b>II</b>) shows a hyperbolic curve plotted between the relative peak current <span class="html-italic">Ip</span> with respect to probe with different concentrations of hybridizing ssGDNA of <span class="html-italic">L. interrogans</span>.</p> "> Figure 4
<p>Comparison of the electrochemical impedance spectra of the DNA chip fabrication steps, including (a) the immobilization of the 5′amino-linked DNA probe and (b–h) hybridization with various concentrations of ssDNA of <span class="html-italic">L. interrogans</span> using 1-mM Potassium ferricyanide solution.</p> "> Figure 5
<p>Cyclic voltametric analysis of DNA sensor selectivity using cDNA (complementary DNA) and a sequence with different numbers of mismatched bases. The insert shows the relative peak current values % <span class="html-italic">Ip</span> (with respect to the probe) of the DNA sensor with cDNA and different numbers of mismatched bases.</p> "> Figure 6
<p>Evaluation of the DNA sensor specificity with <span class="html-italic">L. interrogans</span> and other bacterial species (<span class="html-italic">Escherichia coli, Staphylococcus aureus</span>, and <span class="html-italic">Klebsiella pneumoniae</span>) using cyclic voltametric studies (NC= Negative Control). The insert shows the relative <span class="html-italic">Ip</span> values (with respect to the probe) of the DNA sensor with the hybridizing GDNA of <span class="html-italic">L. interrogans</span> and other bacteria.</p> "> Scheme 1
<p>Illustration of steps involved in the construction of the DNA sensor. AuN: Gold Nanoparticles, CNF: Carbon Nanofiber, SPE: Screen-Printed Electrode, MPA: 3-Mercaptopropionic acid, CV: Cyclic Voltammetry, EIS: Electrochemical Impedance Spectroscopy, EDC: 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide, and NHS: N-Hydroxysuccinimide.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Equipment
2.3. Isolation of Genomic DNA
2.4. Fabrication of the AuN/CNFs DNA Sensor
2.5. Selectivity of the Sensor
3. Results and Discussion
3.1. Characterization Study
3.2. Electrochemical Analysis
3.3. Selectivity of the Sensor
3.4. Specificity Tests
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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DNA Sample | Mismatch Base Sequences |
---|---|
DNA Probe (AmC6) | 5′TCCCGAACAAGCAGAAGGTG3′ |
cDNA | 5′CACCTTCTGCTTGTTCGGGA3′ |
1 BMM | 5′TACCTTCTGCTTGTTCGGGA3′ |
2 BMM | 5′TGCCTTCTGCTTGTTCGGGA3′ |
3 BMM | 5′TGACTTCTGCTTGTTCGGGA3′ |
MBMM | 5′TGAGTTATACTTGGGTGGTC3′ |
SN | Type of Sensor | Biomarker | Nanomaterials | Sensitivity | LOD/ * LOQ | Linearity | Ref. |
---|---|---|---|---|---|---|---|
1 | Amperometric DNA sensor | LipL32 gene | AuN/cMWCNT | 264.5 μA/cm2/ng | 0.015 ng/6 μL | ND | [23] |
2 | Electrochemical immunosensor | rLipL32 protein | SPGE | ND | 0.28 ng/mL * 0.93 ng/mL | 1 to 100 ng/mL | [28] |
3 | Amperometric DNA sensor | LipL32 gene | AuN/cMWCNT | ND | ND | 0.37 to 12 ng/μL | [27] |
4 | Amperometric DNA sensor | Loa22 gene | AuN/CNF | 5431.74 μA/cm2/ng | 0.0077 ng/μL | 0.016 to 67.5 ng/µL | PM |
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Verma, V.; Kala, D.; Gupta, S.; Kumar, H.; Kaushal, A.; Kuča, K.; Cruz-Martins, N.; Kumar, D. Leptospira interrogans Outer Membrane Protein-Based Nanohybrid Sensor for the Diagnosis of Leptospirosis. Sensors 2021, 21, 2552. https://doi.org/10.3390/s21072552
Verma V, Kala D, Gupta S, Kumar H, Kaushal A, Kuča K, Cruz-Martins N, Kumar D. Leptospira interrogans Outer Membrane Protein-Based Nanohybrid Sensor for the Diagnosis of Leptospirosis. Sensors. 2021; 21(7):2552. https://doi.org/10.3390/s21072552
Chicago/Turabian StyleVerma, Vivek, Deepak Kala, Shagun Gupta, Harsh Kumar, Ankur Kaushal, Kamil Kuča, Natália Cruz-Martins, and Dinesh Kumar. 2021. "Leptospira interrogans Outer Membrane Protein-Based Nanohybrid Sensor for the Diagnosis of Leptospirosis" Sensors 21, no. 7: 2552. https://doi.org/10.3390/s21072552
APA StyleVerma, V., Kala, D., Gupta, S., Kumar, H., Kaushal, A., Kuča, K., Cruz-Martins, N., & Kumar, D. (2021). Leptospira interrogans Outer Membrane Protein-Based Nanohybrid Sensor for the Diagnosis of Leptospirosis. Sensors, 21(7), 2552. https://doi.org/10.3390/s21072552